Exogenous retroviruses (RVs) cause neurodegenerative diseases in animals and humans, including human immunodeficiency virus (HIV)-associated neurological disease (HAND), and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Moreover, human endogenous retroviral genes (HERVs) have been implicated in the idiopathic diseases multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), and schizophrenia. Certain murine leukemia viruses (MLVs) cause progressive motor neuron degeneration (MND) that mimics key aspects of human motor neuron diseases, and thus have served as models for understanding the molecular basis of RV-induced neurodegeneration. Prior analysis of MLV MND has shown that it is caused by CNS glial expression of a single retroviral protein, Env. Two major unanswered questions in the field include: How is Env neurotoxic? and In what CNS cell types does Env act to induce neurodegeneration? Prior experiments aimed at identifying infected CNS cell types suggested that we were missing a cell type required for neurodegeneration. In response, we developed a novel strategy that both identifies cryptic CNS RV targets and allows their visualization in live tissue. Our preliminary studies implicate astrocytes as critical for neurodegeneration despite minimal viral protein expression. Therefore, in this grant we proposed to confirm this novel finding in aim 1, and then in aim 2 we will examine the functional relationship between infected astrocytes and physiologically altered neurons. To complement the identification/visualization of astrocyte infection in Aim 1A, Aim 1B will exploit the differential targeting and neurovirulence of ecotropic and amphotropic viruses to confirm that disease requires astrocyte infection. To illustrate the translational potential of this model system to human disease Aim 1C will examine the capacity of HTLV-1 Env/virus to cause neurodegeneration when expressed in murine glia. To explore whether astrocyte Env expression alone is sufficient for neurodegeneration, Aim 1D will examine neuropathology in stem-cell based glial brain chimeras engineered to express MLV and HTLV-1 Envs. In Aim 2, we will address how Env expression in astroglia is toxic to neurons, by assessing neurophysiological changes in 1) astrocytes and 2) neurons with which infected astrocytes interact. These analyses will employ patch clamp electrophysiology and calcium imaging in infected/chimeric brainstem slices. Combined with methodologies for visualizing infected cells, the relationship between Env expressing glia and altered neurons will be established. Because our prior neurophysiologic characterization indicates that glial infection causes rebound neuron hyperexcitability and motor neuron exhaustion, we will initially interrogate astrocyte involvement in calcium dynamics and gliotransmission using tetanus toxin-encoding vectors and doxycycline-regulated dn-SNARE mice. The expectation is that these studies will provide critical new insight into how Env expression in glia induces excitotoxicity, with relevance to developing novel therapy for human neurodegenerative conditions.